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Biomedical subjects

P F Lin

Publications and source records attributed to P F Lin.

At least 19 recordsLinked to original sources
PubMed1991

Brain-derived cells contain a specific binding site for Gp120 which is not the CD4 antigen.

Infection with the human immunodeficiency virus (HIV-1) often produces a set of neuropsychiatric dysfunctions which have been termed the AIDS dementia complex. This complex appears due to the infection of brain cells by HIV-1. If so, brain cells might be expected to contain a binding site for the same viral envelope glycoprotein that enables HIV-1 to bind to other cells (e.g. CD4+ T-cells), gp120. The present study shows that the cells of the brain-derived U-138MG, U-373MG, SK-N-MC and SK-N-SH cell lines bind gp120 in an inhibitable fashion. Binding of gp120 to these cells is inhibited by the dyes Aurintricarboxylic acid (ATA) and Evans blue (EB), which are known to inhibit specific gp120 and HIV-1 binding, and block HIV-1 infection, in CD4-expressing cells. Binding is not inhibited by Aurin, a dye related to ATA but lacking its anti-HIV effects. As expected, anti-CD4 antibodies are ineffective in blocking gp120 binding to brain-derived cells. These results suggest that human brain-derived cells possess a specific binding site for gp120 that is not the CD4 antigen.

Antibodies, Monoclonal↗
PubMed1991

Human interleukin-9: genomic sequence, chromosomal location, and sequences essential for its expression in human T-cell leukemia virus (HTLV)-I-transformed human T cells.

We have isolated the genomic sequence of human interleukin-9 (IL-9) based on its sequence homology with a human IL-9 cDNA isolated from human T-cell leukemia virus (HTLV)-I-transformed T cells by expression cloning. The entire genomic sequence has been determined and the gene consists of five exons and four introns. The human IL-9 gene is mapped to the long arm of human chromosome 5 at band 5q31-32, a region found to be deleted in a number of patients with acquired 5q- abnormalities and hematologic disorders. Several blocks of transcriptional control sequences have been identified at the 5'-flanking region of the human IL-9 gene that may play an important role in the control of IL-9 gene expression. The 5'-regulatory region of the human IL-9 gene also contains sequences identified in the 5'-flanking regions of other cytokine genes mapped to the long arm of human chromosome 5, including IL-3, IL-4, IL-5, and granulocyte-macrophage colony-stimulating factor and other T-cell growth factor genes including IL-2 and IL-6. The IL-9 gene is constitutively expressed in the HTLV-I-transformed human T cells and the expression of IL-9 in these cells can be further induced by 12-O-tetradecanoyl phorbol 13-acetate. Transient transfection analysis using the plasmid containing the 5'-flanking region of IL-9 gene upstream from the firefly luciferase ciferase report gene indicated that the 0.9-kb Smal-Sacl fragment of the IL-9 gene contains sequences required for the constitutive and activated expression of IL-9 gene in HTLV-I-transformed cells. These results will now allow us to study the regulatory mechanism of IL-9 gene expression in normal and leukemic human T cells.

Amino Acid Sequence↗
PubMed1991

Growth modulation by epidermal growth factor (EGF) in human colonic carcinoma cells: constitutive expression of the human EGF gene.

The functional role of epidermal growth factor (EGF) in epithelium-derived human colonic carcinoma cells was investigated by transfection with plasmid pUCDS3, which contained synthetic human EGF encoding sequences, into two human colonic carcinoma cell types with dissimilar phenotypic properties: the moderately differentiated and growth factor-responsive Moser and the highly metastatic KM12SM cells. The Moser cells exhibited a proliferative response to treatment with exogenous EGF, while the KM12SM cells did not. The constitutive expression of the human EGF gene in these colonic carcinoma cell types resulted in elevated expression of EGF mRNA, with concurrent production and secretion of a large amount of EGF, and downmodulation of transforming growth factor-alpha (TGF-alpha) secretion. Growth stimulation and down-modulation of both high and low affinity EGF receptors were observed in the EGF-transfected Moser clones. Results of experiments using anti-EGF and anti-EGF-receptor antibody to block the proliferation of EGF-transfected Moser clones suggested that autocrine stimulatory mechanisms involving both EGF and TGF-alpha were operative in these cells. By comparison, a growth-inhibitory effect, with no apparent EGF receptor modulation, was observed in the EGF-transfected KM12SM clones. Both the parental and EGF-transfected KM12SM clones possessed fewer EGF receptors than the Moser cells, and anti-EGF or anti-EGF-receptor antibody did not affect the cells' growth properties. These results suggested that the mechanisms of growth inhibition in the EGF-transfected KM12SM clones were non-autocrine or intracellular in nature. Thus, constitutive expression of the human EGF gene in two phenotypically different, epithelium-derived human colonic carcinoma cells resulted in divergent altered growth characteristics.

Cell Division↗
PubMed1990

Molecular dissection of mutations at the heterozygous thymidine kinase locus in mouse lymphoma cells.

The mouse lymphoma L5178Y TK+/- 3.7.2C cell line allows quantitation of induced TK(+/-)----TK-/- mutations at the heterozygous thymidine kinase (Tk) locus. TK-/- mutant colonies show a bimodal size distribution, reflecting a difference in the growth rates of the two size classes that is hypothesized to result from different degrees of genetic damage. The two homologous chromosomes 11 containing the alleles of the Tk gene in L5178Y 3.7.2C TK+/- cells are distinguishable at the cytogenetic level. We find, in addition, that the two alleles are distinguishable at the molecular level because of an Nco I restriction fragment length polymorphism at the 3' end of the gene. In a set of 51 large-colony and 48 small-colony TK-/- mutants induced by ionizing radiation or by chemical mutagens, we find that 78, including all except one of the small-colony mutants, have lost the Tk+ allele and that some of these have two to four copies of the remaining Tk- allele. Nineteen of the large-colony TK-/- mutants that do not show Tk+ allele loss show no other structural changes detectable at the level of Southern blot analysis. One shows a partial deletion. The variety of mutagenic lesions recorded at the heterozygous Tk locus may be representative of events observed in human malignancy and may include a wider range of mutagenic events than can be observed at hemizygous test loci.

Alleles↗
PubMed1989

Protection of mice against Listeria monocytogenes infection by recombinant human tumor necrosis factor alpha.

Recombinant human tumor necrosis factor alpha (rHuTNF-alpha) administered intravenously to mice resulted in enhanced resistance to a lethal challenge infection of Listeria monocytogenes given 24 h later. The observed protection was lost following treatment of the rHuTNF-alpha preparations with rabbit polyclonal antibody rHuTNF-alpha but not with normal rabbit immunoglobulin G.

Animals↗
PubMed1988

Induction of tumor necrosis factor, IFN-gamma, and acute lethality in mice by toxic and non-toxic forms of lipid A.

The effect of the i.v. administration of endotoxin (LPS), diphosphoryl lipid A, and the non-toxic derivative monophosphoryl lipid A (MPL), on the production of serum cachectin (TNF), IFN-gamma, and the appearance of endotoxin shock have been measured in mice primed with Listeria monocytogenes. All three of the lipid A varieties induced the production of TNF in a dose-dependent manner. Although comparable peak levels of TNF were produced (5 X 10(5) to 1 X 10(6) U/ml), treatments with LPS or diphosphoryl lipid A were lethal whereas those with MPL were not. A study following TNF in the mouse sera for up to 24 h after treatment with the lipid A types showed that serum levels of TNF peaked 90 min after the treatment, and that TNF levels induced by LPS treatment were maintained for several hours longer than those induced by lipid A or MPL. All three molecules also resulted in the production of IFN-gamma in the serum, which peaked at 4 to 5 h after treatment. After 90 min there were no significant differences in the levels of serum IFN-gamma in any of the groups of treated animals. However, as was observed with the TNF, the levels of IFN in animals treated with LPS persisted longer than those induced by MPL or lipid A. These results suggest that the non-toxic MPL as well as the toxic forms of lipid A can induce the production of TNF by macrophages. Furthermore, although it is essential, TNF alone is not necessarily sufficient to induce septic shock in mice.

Adjuvants, Immunologic↗
PubMed1988

Transcriptional and posttranscriptional mechanisms regulate murine thymidine kinase gene expression in serum-stimulated cells.

We previously isolated and characterized the structure of murine thymidine kinase (tk) genomic and cDNA sequences to begin a study designed to identify regions of the tk gene important for regulated expression during the transition of cells from G0 to a proliferating state. In this report, we describe the stable transfection of the cloned gene into L-M(TK-) cells and show that both thymidine kinase (TK) enzyme activity and DNA synthesis increase in parallel when transfectants in G0 arrest are stimulated by serum. To define promoter and regulatory regions more precisely, we have constructed a series of tk minigenes and have examined their expression in stable transfectants after serum stimulation. We have identified a 291-base-pair DNA fragment at the 5' end of the tk gene that has promoter function, and we have determined its sequence. In addition, we have found that DNA sequences which mediate serum-induced expression of TK are transcribed, since expression of the murine tk cDNA, fused to a promoter from either the murine tk gene, the simian virus 40 early region, or the herpes simplex virus tk gene, is stimulated by serum. Our constructs also reveal that the murine tk polyadenylation signal is not required for regulation, nor is most of the 3' untranslated region. RNA dot blot analysis indicates that murine cytoplasmic tk mRNA levels always parallel TK enzyme activity. Nuclear runon transcription assays show less than a 2-fold increase in transcription from the cloned tk gene in serum-stimulated transfectants, but an 11-fold increase in mouse L929 cells, which are inherently TK+. These results taken together suggest that the murine tk gene is controlled in serum-stimulated cells by a transcriptional mechanism influenced by DNA sequences that flank tk and also by a posttranscriptional system linked to gene sequences that are transcribed.

Animals↗
PubMed1988

[Antitumor effect of actinidia chinensis polysaccharide on murine tumor].

A new polysaccharide compound (ACPS-R) has recently been isolated from the root of Actinidia Chinensis Planch. When given intraperitoneally to the transplantable tumor bearing mice at dose of 75-125 mg/kg, the tumor inhibition rate was more than 88.8% in Ehrilich ascitic cancer (EAC) or ascitic form of hepatoma (HepA) and more than 49.6% in solid hepatoma (HepS). The treatment effect of ACPS-R on EAC at dose of 15 mg/kg and 22.5 mg/kg, respectively. ACPS-R could also prolong the life of EAC-or P388-bearing mice, and increase the percentage of EAC-free mice. In addition, when ACPS-R was used in combination with 5-Fu, the antitumor effect was enhanced as compared with 5-Fu alone. A marked increase in cAMP levels and cAMP/cGMP ratio of spleen of EAC-bearing mice were observed after treatment of ACPS-R. The increase of both parameters nearly reached the normal levels of healthy mice. The increases of cAMP, cAMP/cGMP and tumor remission had statistical significance. It showed an intermediate inhibitory effect of ACPS-R on DNA synthesis by incorporating 3H-TdR into EAC cells. The results indicated that ACPS-R acts as a new antitumor polysaccharide, and the treatment effect of Actinidia root in folk medicine is probably related to ACPS-R.

Animals↗
PubMed1987

Cloning and expression of murine lymphotoxin cDNA.

The murine lymphotoxin (LT) gene has been cloned and used to identify cDNA clones in a library prepared from activated murine T cell mRNA. A recombinant murine genomic library was screened with a human lymphotoxin cDNA probe, resulting in the isolation of the entire LT gene. The murine LT gene structure is similar to the human gene, containing three intervening sequences. An activated murine T cell cDNA library was prepared with poly(A)+ RNA isolated 7 hr after concanavalin A stimulation of an L3T4+ interleukin 2-dependent murine T cell clone. Two colonies of the cDNA library that contained inserts that hybridized with the murine LT gene probe were sequenced and were used to construct expression plasmids. The amino acid sequence deduced from the cDNA indicates that murine LT is highly homologous to human LT (74%) and is related to murine tumor necrosis factor (35% homology). The cDNA was transcribed and was translated in vitro, and was expressed in COS-1 cells. This has resulted in the production of LT biological activity.

Amino Acid Sequence↗
PubMed1985

Molecular cloning and structural analysis of murine thymidine kinase genomic and cDNA sequences.

Two functional cytosolic thymidine kinase (tk) cDNA clones were isolated from a mouse L-cell library. An RNA blot analysis indicated that one of these clones contains a nearly full-length tk sequence and that LTK- cells contain little or no TK message. The nucleotide sequences of both clones were determined, and the functional mouse tk cDNA contains 1,156 base pairs. An analysis of the sequence implied that there is an untranslated 32-nucleotide region at the 5' end of the mRNA, followed by an open reading frame of 699 nucleotides. The 3' untranslated region is 422 nucleotides long. Thus, the gene codes for a protein containing 233 amino acids, with a molecular weight of 25,873. A comparison of the coding sequences of the mouse tk cDNA with the human and chicken tk genes revealed about 86 and 70% homology, respectively. We also isolated the tk gene from a mouse C57BL/10J cosmid library. The structural organization was determined by restriction mapping, Southern blotting, and heteroduplex analysis of the cloned sequences, in combination with a mouse tk cDNA. The tk gene spans approximately 11 kilobases and contains at least five introns. Southern blot analysis revealed that this gene is deleted in mouse LTK- cells, consistent with the inability of these cells to synthesize TK message. This analysis also showed that tk-related sequences are present in the genomes of several mouse strains, as well as in LTK- cells. These segments may represent pseudogenes.

Amino Acid Sequence↗
PubMed1979

Genetic control of cobalamin binding in normal and mutant cells: assignment of the gene for 5-methyltetrahydrofolate:L-homocysteine S-methyltransferase to human chromosome 1.

When extracts prepared from cultured human or rodent fibroblasts grown in medium containing [(57)Co]cobalamin were analyzed by polyacrylamide gel electrophoresis, most of the intracellular radioactivity migrated with the activity of the cobalamin-dependent enzyme 5-methyltetrahydrofolate:L-homocysteine S-methyltransferase (EC 2.1.1.13). Because the rodent and human forms of this enzyme are electrophoretically different, we used the binding of [(57)Co]cobalamin to detect the presence of the human methyltransferase isozyme in rodent-human somatic cell hybrids. As expected, binding and methyltransferase activities were found to cosegregate, thus confirming genetically their electrophoretic identity. Accordingly, we examined the [(57)Co]cobalamin-binding patterns and human chromosome contents of a panel of 12 rodent-human hybrid clones, and concluded that the gene for the methyltransferase (designated Mtr) is located on human chromosome 1. Using this information, we probed the nature of the molecular defect exhibited by fibroblasts cultured from patients expressing the cbl C mutation. Although these cells are unable to associate newly taken up [(57)Co]cobalamin with the methyltransferase, hybrids of mouse L-cells and cbl C cells containing chromosome 1 show a "reappearance" of the human [(57)Co]cobalamin-methyltransferase. These results indicate that the cbl C mutation does not affect the methyltransferase apoprotein, but rather some metabolic step that must convert cobalamin to a chemical form capable of attaching to the enzyme.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1977

Initiation of genetic exchanges in lambda phage--prophage crosses.

When Escherichia coli K-12 (lambda) lysogens were infected with lambda phages, genetic exchanges between phage and prophage occurred at low frequencies (less than 0.1% between the markers P3 and P80), but at frequencies above 1% if the infecting phages were first treated with the photosensitizing agent 4,5',8-trimethylpsoralen and 360 nm light. Exchanges were induced by psoralen damage at about the same frequency in wild-type lysogens and in those carrying recB(-), recC(-), recF(-), or lexA(-), but at an intermediate frequency in a quadruple mutant carrying recB(-)recC(-)recF(-)sbcB(-). Few if any exchanges were induced in lysogens carrying uvrA(-), uvrB(-), or recA(-). The increase in the frequency of recombination was presumably due to the psoralen damage in the phage DNA molecules and the action of host cell repair and recombination enzymes. The production of crosslinks in the phage DNA by psoralen and 360 nm light was measured by sedimentation in alkali. It showed second-order kinetics indicative of a two-photon reaction. In contrast, first-order kinetics had been reported for monoadduct formation. Second-order kinetics, similar to those for crosslink production, were found for genetic exchanges in homoimmune crosses. Presumably, crosslinks, rather than monoadducts, cause most of the exchanges. Because the uvrA(-) gene product (UV-endonuclease) was required, it is likely that recombination was initiated by DNA molecules cut at crosslinks. This system has been used to show that after the crosslinked phage duplex has been cut, one or more of the subsequent steps-homologous pairing, cutting, and joining-require the recA(+) gene product.

Coliphages↗
PubMed1976

Genetic exchanges caused by ultraviolet photoproducts in phage lambda DNA molecules: the role of DNA replication.

Genetic recombination induced by structural damage in DNA molecules was investigated in E. coli K12 (lambda) lysogens infected with genetically marked phage lambda. Photoproducts were induced in the phage DNA before infection by exposing them either to 313 nm light in the presence of acetophenone or to 254 nm light. To test the role of the replication of the damaged phage DNA on the frequency of the induced recombination, both heteroimmune and homimmune crosses were performed. First, samples of a heteroimmune phage lambda imm434 P80 exposed to these treatments were allowed to infect cells lysogenic for prophage lambda cI857 P3. Phage DNA replication and maturation took place, and the resulting progeny phages were assayed for the frequency of P+ recombinants. Recombination was less frequent in infected cells exposed to visible light and in wild type cells able to perform excision repair than in excision-defective lysogens. Therefore, much of the induced recombination can be attributed to the pyrimidine dimers in the phage DNA, the only photoproducts known to be dissociated by photoreactivating enzyme. Second, in homoimmune crosses, samples of similarly treated homoimmune lambda P3 phages were allowed to infect lysogens carrying lambda cI857 P80. Replication of the phage DNA containing ultraviolet photoproducts was repressed by lambda immunity, and was further blocked by the lack of the P gene product needed for replication. The lysogens were purified and scored for both colony forming ability and for P+ recombinant prophages. The 254 nm photoproducts increased the frequency of recombination in these homimmune crosses, even though phage DNA replication was blocked. Irradiation with 313 nm light and acetophenone M, which produces dimers and unknown photoproducts, was not as effective per dimer as the 254 nm light. It is concluded from these results that certain unidentified 254 nm photoproducts can cause recombination even in the absence of DNA replication. They are not pyrimidine dimers, as they are not susceptible to excision repair or photoreactivation. In contrast, pyrimidine dimers appear to cause recombination only when the DNA containing them undergoes replication.

Coliphages↗
PubMed1975

Genetic exchanges induced by structural damage in nonreplicating phage lambda DNA.

Genetic recombination between irradiated lambda phage and the unirradiated lambda prophage in homoimmune lysogens has been studied under conditions in which phage DNA replication and repair were controlled. The lambda phage were exposed to one of three treatments before infecting the lysogens: (a) 254-nm light, which produces pyrimidine dimers and other photoproducts; (b) 313-nm light with acetopheneone D, which produces thymine dimers and a different spectrum of other photoproducts; (c) 360-nm light with trimethylpsoralen, which produces monoadducts and cross-links. With both replication and excision-repair of the damaged phage DNA blocked, treatment b (acetophenone D) caused no significant increase in recombination, indicating that thymine dimers do not cause recombination if the DNA in which they are contained is not replicated. Treatment a (254 nm), producing the same total number of pyrimidine dimers, caused a marked increase in recombination. This indicates that photoproducts other than pyrimidine dimers produced by 254-nm light can cause recombination in the absence of replication. Treatment c (psoralen) caused a marked increase in recombination in wild type but not in uvrA and uvrB mutants. The frequency of recombination in two-factor crosses varied with marker separation in such a way as to suggest that cross-links can act over distances of at least 5% of the lambda genome to cause exchanges between pairs of relatively closely spaced markers. The psoralen photo cross-links and monoadducts initiate recombination only following the action of excision enzymes, which appears to release one arm of each cross-link, producing a gap with free strand ends. It may be these strand ends which induce recombination. The action at a distance of 5% of the lambda genome may reflect heteroduplex formation and the subsequent reduction to homozygosity of mismatched base pairs at genetic markers. Recombination between closely spaced markers in the P gene is reduced in strains carrying polA.

Coliphages↗